Inhibitors of alpha-tubulin acetylation for the treatment of pain

ABSTRACT

The present invention pertains to novel analgesics useful for treating mechanical pain. The invention suggests the use of inhibitors of α-tubulin acetylation for inhibition of neurological sensations that are mediated by sensory neurons. The perception of mechanical pain is can be modulated by altering the α-tubulin acetylation, in context of the invention in particular by modulation of the expression and/or activity of the enzyme α-tubulin acetyltransferase (Atat). The invention provides the medical application of α-tubulin acetyltransferase inhibitors as analgesics and a screening method for the identification of compounds useful in the treatment of pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/069,818, now granted as U.S. Pat. No. 11,104,949, filed Jul. 12,2018, which is the National Stage under 35 USC § 371 of InternationalApplication No. PCT/EP2016/082765, filed Dec. 28, 2016, which claimspriority to European Application No. 16151345.2, filed Jan. 14, 2016.

FIELD OF THE INVENTION

The present invention pertains to novel analgesics useful for treatingmechanical pain. The invention suggests the use of inhibitors ofα-tubulin acetylation for inhibition of neuro-logical sensations thatare mediated by sensory neurons. The perception of mechanical pain iscan be modulated by altering the α-tubulin acetylation, in context ofthe invention in particular by modulation of the expression and/oractivity of the enzyme α-tubulin acetyltransferase (Atat). The inventionprovides the medical application of α-tubulin acetyltransferaseinhibitors as analgesics and a screening method for the identificationof compounds useful in the treatment of pain.

DESCRIPTION

Pain is a complex subjective sensation reflecting real or potentialtissue damage and the affective response to it. Acute pain is aphysiological signal indicating a potential or actual injury. Chronicpain can either be somatogenetic (organic) or psychogenic. Chronic painis frequently accompanied or followed by vegetative signs, which oftenresult in depression. Chronic pain results in individual suffering andsocial economic costs of tremendous extent. Existing pharmacologicalpain therapies are widely unsatisfying both in terms of efficacy and ofsafety. Somatogenetic pain may be of nociceptive origin, inflammatory orneuropathic. Nociceptive pain is judged to be commensurate with ongoingactivation of somatic or visceral pain-sensitive nerve fibers.Neuropathic pain results from dysfunction in the nervous system that issustained by aberrant somatosensory processes in the peripheral nervoussystem.

Neuropathic pain is a persistent or chronic pain syndrome that canresult from damage to the nervous system, the peripheral nerves, thedorsal root ganglion, dorsal root, or to the central nervous system.Neuropathic pain syndromes include allodynia, various neuralgias such aspost herpetic neuralgia and trigeminal neuralgia, phantom pain, andcomplex regional pain syndromes, such as reflex sympathetic dystrophyand causalgia. Causalgia is often characterized by spontaneous burningpain combined with hyperalgesia and allodynia. Tragically there is noexisting method for adequately, predictably and specifically treatingestablished neuropathic pain as present treatment methods forneuropathic pain consists of merely trying to help the patient copethrough psychological or occupational therapy, rather than by reducingor eliminating the pain experienced. Treatment of neuropathic or chronicpain is a challenge for physicians and patients since there are nomedications that specifically target the condition, and since themedications presently used result in only little relief and are based ontheir efficacy in acute pain conditions or on their efficacy onrelieving secondary effects like anxiety and depression. Incidence ofchronic pain is increasing in society and its burden on society is hugein both health care and lost productivity. Currently there are noscientifically validated therapies for relieving chronic pain. As aresult, the health community targets ‘pain management’ where multi-modaltherapies are used concurrently with the hope of providing someimprovement in quality of life. Thus, there is an urgent need for drugsthat can relieve chronic pain.

Pain is often caused by mechanical stimuli. Mechanical forces actingupon cells or tissues can be propagated into the opening of mechanicallygated ion channels either through direct interplay of ion channels withthe lipid bilayer or through further interaction with other cellularcomponents such as the underlying cytoskeleton. The bacterialmechanosensitive channel MscS7 and eukaryotic two-pore-domain potassiumchannels TRAAK and TREK18 are fully activated by mechanical stimuli whenreconstituted in reduced membrane systems indicating that theirmechanosensitivity is a result of interactions with the plasma membrane.However, from in vivo experiments carried out in eukaryotes there isevidence that mechanotransduction depends on further cellular componentsto amplify and shape mechanical sensitivity.

Hence, until this day there is no specific therapy for mechanical painavailable. The present invention seeks to provide novel analgesics thataddress problems of state of the art pain treatments, such as highlytoxic side effects, and particularly target pain sensation induced bymechanical stimuli.

The above problem is solved in a first aspect by an inhibitor ofα-tubulin acetylation for use in the inhibition of a neurologicalsensation in a subject, wherein the neurological sensation is mediatedby sensory neurons.

As used herein, the term “sensory neurons” relates to neurons configuredto transmit neural stimuli corresponding to sensory stimuli. Accordingto some embodiments, sensory neurons are activated by physical and/orchemical stimuli, such as, but not limited to, mechano-sensors and/orchemo-receptors. Preferred are sensory neurons which are dorsal rootganglion (DRG) neurons.

As used herein, the term “acetylation” refers to an enzymatic transferof acetyl groups from donor molecules (e.g., acetyl CoA) to specifictarget substrates. In context of the present invention the preferredtarget molecule of the enzymatic transfer is tubulin, more preferablyα-tubulin. Acetylation of the ε-amino group of lysine 40 on α-tubulin isa conserved post translational modification on the luminal side ofmicrotubules (Nogales et al., 1999 Cell 96:79-88) that was discovered inthe flagella of Chlamydomonas reinhardtii (L'Hernault and Rosenbaum,1983 J. Cell Biol. 97:258-263; LeDizet and Piperno, 1987 Proc. Natl.Acad. Sci. USA 84:5720-5724). Studies on the significance of microtubuleacetylation have been limited so far.

In context of the present invention it was surprisingly shown thatdeletion of Atat1, the main enzyme responsible for α-tubulin acetylationin mammals, from peripheral sensory neurons results in a profound andremarkably selective loss on mechanical sensation in mice. The inventorsdemonstrate that this impacts upon both light touch and pain, and thatall mechano-receptor subtypes which innervate the skin are lessresponsive in the absence of α-tubulin acetylation. The inventorspropose that this phenotype is caused by an increased mechanicalstiffness of sensory neurons that is in turn mediated by the loss of asub-membrane band of acetylated α-tubulin in Atat1^(cKO) mice.Modulation of α-tubulin acetylation in neuronal cells is therefore a newapproach to modify the sensation of tactile and pain stimuli, and othermechanically induced perceptions.

The term “neurological sensation” in context of the invention has to beunderstood to refer to any perception of in particular mechanicalstimuli by an animal or human, most preferably tactile sensations(touch) and/or pain. Further included is the identical perception thatoccurs without the presence of a mechanical stimulus in the event of afor example pathologic chronic perception of the neurologicalsensation—such as chronic pain. Preferably the neurological sensation ismediated by peripheral sensory neurons, most preferably which are DRGperipheral sensory neurons.

The term “pain” as used herein refers to an unpleasant sensation. Forexample, the subject experiences discomfort, distress or suffering. Itis known to one skilled in the art that various painful conditions maybe classified according to broadly opposing or otherwise usefulcategories. Examples of opposing categories include; nociceptive painversus non-nociceptive pain, and acute pain versus chronic pain.Examples of other common categories of pain used by those skilled in theart include mechanical pain, neuropathic pain and phantom pain.

As used herein, the term “mechanical pain” refers to pain other thanheadache pain that is not neuropathic or a result of exposure to heat,cold or external chemical stimuli. Mechanical pain includes physicaltrauma (other than thermal or chemical burns or other irritating and/orpainful exposures to noxious chemicals) such as postsurgical pain andpain from cuts, bruises and broken bones; toothache, denture pain; nerveroot pain; osteoarthritis; rheumatoid arthritis; fibromyalgia; meralgiaparesthetica; back pain; cancer-associated pain; angina; carpel tunnelsyndrome; and pain resulting from bone fracture, labor, hemorrhoids,intestinal gas, dyspepsia, and menstruation. Itching conditions that maybe treated include psoriatic pruritis, itch due to hemodialysis,aguagenic pruritus, and itching associated with vulvar vestibulitis,contact dermatitis, insect bites and skin allergies.

The term “nociception” as used herein refers to the transduction ofnoxious or potentially injurious stimuli into a sensation.

The term “nociceptive pain” as used herein refers to pain caused byactivity in primary sensory pain fibers in the peripheral nervoussystem. Neurons in the peripheral nervous system that typically respondto noxious or painful stimuli are commonly referred to as nociceptors ornociceptive neurons. Yet further, the nociceoptive pathways extend tothe somatosensory cortex.

The term “non-nociceptive pain” as used herein refers to pain caused byactivity in neurons in the central nervous system. Examples of neuronsin the central nervous system that may cause non-nociceptive paininclude neurons in the dorsal horn of the spinal cord such asinterneurons and projection neurons, or neurons in parts of the brainknown to be involved in pain sensation such as the rostral ventromedialmedulla (RVM) and the periaqueductal grey (PAG).

The term “acute pain” as used herein refers to pain that is transient innature or lasting less than 1 month. Acute pain is typically associatedwith an immediate injurious process such as soft tissue damage,infection, or inflammation, and serves the purpose of notifying thesubject of the injurious condition, thus allowing for treatment andprevention of further injury.

The term “chronic pain” as used herein refers to pain that lasts longerthan 1 month or beyond the resolution of an acute tissue injury or isrecurring or is associated with tissue injury and/or chronic diseasesthat are expected to continue or progress. Examples of chronic diseasesthat are expected to continue or progress may include cancer, arthritis,inflammatory disease, chronic wounds, cardiovascular accidents, spinalcord disorders, central nervous system disorder or recovery fromsurgery.

The term “neuropathy” as used herein refers to any condition thatadversely affects the normal functioning of the nervous system.Neuropathies can originate anywhere in the central or peripheral nervoussystem, but only in some cases does this produce neuropathic pain.

The term “neuropathic pain” as used herein refers to pain that resultfrom damage to or abnormal function of the nervous system itself. It mayexist independently of any form of tissue injury outside of the nervoussystem. Examples of conditions that may lead to neuropathic pain includedisease (e.g., HIV, Herpes, Diabetes, Cancer, autoimmune disorders),acute trauma (surgery, injury, electric shock), and chronic trauma(repetitive motion disorders, chemical toxicity such as alcohol,chemotherapy, or heavy metals).

The term “phantom pain” as used herein refers to a condition whereby thepatient senses pain in a part of the body that is either no longerphysically present due to amputation, or is known to be completelyinsensate due to total peripheral nerve destruction.

The term “hyperalgesia” as used herein refers to an increasedsensitivity to nociceptive or painful stimuli. The term “allodynia” asused herein describes a condition whereby normally non-noxious stimuliare perceived as painful. Both hyperalgesia and allodynia can be dividedinto primary and secondary categories or conditions. Primaryhyperalgesia/allodynia is an increase in sensitivity to painful andpreviously non-painful stimuli in a region of the body that hasundergone tissue injury. Secondary hyperalgesia/allodynia is an increasein pain sensitivity globally and requires descending input into theperiphery from various pain processing centers in the brain.

In accordance with the invention alternative embodiments also pertain toa sensory neuron mediated sensation which is, itching of the skin, aburning sensation, or a nociceptive (pain) sensation. As alreadymentioned above, the pain can be selected from inflammatory pain,inflammatory hyperalgesia, hyperalgesia, neuropathic pain, migraine,cancer pain, visceral pain, osteoarthritis pain, chronic pain andpost-surgical pain. However, neuropathic pain is most preferred.

An inhibitor of α-tubulin acetylation in context of the invention may beany compound that impairs or interferes with the enzymatic transfer ofan acetyl group from a donor molecule to α-tubulin. Preferred are, incontext of the invention, such inhibitors that inhibit α-tubulinacetylation specifically and selectively in neuronal cells, such asperipheral sensory neurons. In one embodiment, the inhibitor ofα-tubulin acetylation may inhibit α-tubulin acetylation via a directinteraction with the α-tubulin acetyltransferase, its RNA transcript orits coding genetic locus. Such inhibitors in context of the inventionwill be referred to as “α-tubulin acetyltransferase inhibitors orantagonists”, or similar expressions. In other embodiments, theinvention also includes inhibitors of α-tubulin acetylation thatinteract with other components of the acetylation reaction, for examplewith α-tubulin (the substrate) or the acetyl donor molecule. One exampleof such an inhibitor would be a genetic construct mediating a e.g.CRISPR/Cas9 gene editing of α-tubulin to create a K40 α-tubulin mutantthat loses the ability of acting as an acceptor of an acetyl moiety.

As mentioned earlier, preferred embodiments of the invention pertain toα-tubulin acetyltransferase inhibitors or antagonists as inhibitors ofα-tubulin acetylation. More preferably the α-tubulin acetyltransferaseinhibitor or antagonist is an inhibitor or antagonist of mammalian/humanAtat1.

The inhibitor of α-tubulin acetylation of the invention is preferablyfor use in medicine. Therefore, the inhibition of a neurologicalsensation in a subject may be a prevention or treatment of a noxiousneurological sensation in the subject, and preferably is the preventionand/or treatment of pain, as defined herein above.

A prevention or treatment of pain in context of the herein disclosedinvention preferably comprises the administration of the inhibitor tothe subject suspected to suffer from, or suffering from pain. Painpreferably is selected from the group including acute mechanical pain,chronic mechanical pain, mechanical hyperalgesia, mechanical allodynia,inflammation, dental pain, cancer pain, visceral pain, arthritis pain,post-surgical pain, neuropathic pain, and labor pain.

In context of the present invention the term “subject” preferably refersto a mammal, preferably a human. The subject of the invention may be atdanger of suffering from pain, or suffer from pain, wherein the pain isas defined herein above.

The inhibitor or antagonist of α-tubulin acetylation of the invention isin some embodiments selected from a compound having an inhibitoryactivity towards α-tubulin acetyltransferase and which is a polypeptide,peptide, glycoprotein, a peptidomimetic, an antibody or antibody-likemolecules; a nucleic acid such as a DNA or RNA, for example an antisenseDNA or RNA, a ribozyme, an RNA or DNA aptamer, siRNA and the like,including variants or derivatives thereof such as a peptide nucleic acid(PNA); a carbohydrate such as a polysaccharide or oligosaccharide andthe like, including variants or derivatives thereof; a lipid such as afatty acid and the like, including variants or derivatives thereof; or asmall organic molecules including but not limited to small moleculeligands, small cell-permeable molecules, and peptidomimetic compounds.

The person of skill in the pertinent art is well aware of options of howto inhibit α-tubulin acetylation in a neuronal cell. Inhibitors ofα-tubulin acetylation are for example disclosed and described in US2014/0248635, which is incorporated by reference in its entirety.

As used herein, the term “α-tubulin acetyltransferase antagonist orinhibitor” means a substance that affects a decrease in the amount orrate of α-tubulin acetyltransferase expression or activity. Such asubstance can act directly, for example, by binding to α-tubulinacetyltransferase and decreasing the amount or rate of α-tubulinacetyltransferase expression or, in particular, its enzymatic activity.A α-tubulin acetyltransferase antagonist or inhibitor can also decreasethe amount or rate of α-tubulin acetyltransferase expression oractivity, for example, by binding to α-tubulin acetyltransferase in sucha way as to reduce or prevent interaction of α-tubulin acetyltransferasewith its substrate; by binding to α-tubulin acetyltransferase andmodifying it, such as by removal or addition of a moiety, or alteringits three-dimensional conformation; and by binding to α-tubulinacetyltransferase and reducing its stability or conformationalintegrity. A α-tubulin acetyltransferase antagonist or inhibitor canalso act indirectly, for example, by binding to a regulatory molecule orgene region so as to modulate regulatory protein or gene region functionand affect a decrease in the amount or rate of α-tubulinacetyltransferase expression or activity. Thus, a α-tubulinacetyltransferase inhibitor or antagonist can act by any mechanisms thatresult in a decrease in the amount or rate of α-tubulinacetyltransferase expression or activity.

An α-tubulin acetyltransferase antagonist or inhibitor can be, forexample, a naturally or non-naturally occurring macromolecule, such as apolypeptide, peptide, peptidomimetic, nucleic acid, carbohydrate orlipid. An α-tubulin acetyltransferase antagonist or inhibitor furthercan be an antibody, or antigen-binding fragment thereof, such as amonoclonal antibody, humanized antibody, chimeric antibody, minibody,bifunctional anti-body, single chain antibody (scFv), variable regionfragment (Fv or Fd), Fab or F(ab)2. A α-tubulin acetyltransferaseantagonist or inhibitor can also be polyclonal antibodies specific forα-tubulin acetyltransferase. A α-tubulin acetyltransferase antagonist orinhibitor further can be a partially or completely synthetic derivative,analog or mimetic of a naturally occurring macromolecule, or a smallorganic or inorganic molecule.

A α-tubulin acetyltransferase antagonist or inhibitor that is anantibody can be, for example, an antibody that binds to α-tubulinacetyltransferase and inhibits binding of a substrate to α-tubulinacetyltransferase, such as a binding to tubulin, or alters the activityof a molecule that regulates α-tubulin acetyltransferase expression oractivity, such that the amount or rate of α-tubulin acetyltransferaseexpression or activity is decreased. An antibody useful in a method ofthe invention can be a naturally occurring antibody, includingmonoclonal or polyclonal antibodies or fragments thereof, or anon-naturally occurring antibody, including but not limited to a singlechain antibody, chimeric antibody, bifunctional antibody,complementarity determining region-grafted (CDR-grafted) antibody andhumanized antibody or an antigen-binding fragment thereof.

An α-tubulin acetyltransferase antagonist or inhibitor that is a nucleicacid can be, for example, an anti-sense nucleotide sequence, an RNAmolecule, or an aptamer sequence. An anti-sense nucleotide sequence canbind to a nucleotide sequence within a cell and modulate the level ofexpression of α-tubulin acetyltransferase or modulate expression ofanother gene that controls the expression or activity of an α-tubulinacetyltransferase. Similarly, an RNA molecule, such as a catalyticribozyme, can bind to and alter the expression of the α-tubulinacetyltransferase gene, or other gene that controls the expression oractivity of α-tubulin acetyltransferase. An aptamer is a nucleic acidsequence that has a three dimensional structure capable of binding to amolecular target.

Certain preferred embodiments pertain to genetic constructs for geneediting that are used as inhibitors of α-tubulin acetylation in contextof the herein described invention. By using gene editing it is possibleto modulate the expression, stability or activity of the α-tubulinacetyltransferase. Alternatively, gene editing may be used to alterα-tubulin itself in order to reduce α-tubulin acetylation, for exampleby mutating lysine 40 which is a known target site for α-tubulinacetylation. Gene editing approaches are well known in the art and maybe easily applied when the target gene sequences are known. Preferablysuch approaches may be used in gene therapy using viral vectors andwhich specifically target sensory neurons in accordance with the abovedescriptions.

An α-tubulin acetyltransferase antagonist or inhibitor that is a nucleicacid also can be a double-stranded RNA molecule for use in RNAinterference methods. RNA interference (RNAi) is a process ofsequence-specific gene silencing by post-transcriptional RNA degradationor silencing. The RNAi is initiated by use of double-stranded RNA(dsRNA) that is homologous in sequence to the target gene to besilenced. A suitable double-stranded RNA (dsRNA) for RNAi contains senseand antisense strands of about 21 contiguous nucleotides correspondingto the gene to be targeted that form 19 RNA base pairs, leavingoverhangs of two nucleotides at each 3′ end (Elbashir et al., Nature411:494-498 (2001); Bass, Nature 411:428-429 (2001); Zamore, Nat.Struct. Biol. 8:746-750 (2001)). dsRNAs of about 25-30 nucleotides havealso been used successfully for RNAi (Karabinos et al., Proc. Natl.Acad. Sci. USA 98:7863-7868 (2001). dsRNA can be synthesized in vitroand introduced into a cell by methods known in the art.

Within certain preferred aspects, alpha tubulin acetylation modulatorsas provided herein may be used for the treatment of mechanical pain.

Another aspect of the present invention pertains to a pharmaceuticalcomposition for use in the prevention or treatment of pain. Thepharmaceutical composition of the invention comprises an inhibitor ofα-tubulin acetylation as described herein above, and a pharmaceuticalacceptable carrier and/or excipient.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, solubilizers, fillers,stabilizers, binders, absorbents, bases, buffering agents, lubricants,controlled release vehicles, diluents, emulsifying agents, humectants,lubricants, dispersion media, coatings, antibacterial or antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well-known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary agents can also be incorporated into the compositions. Incertain embodiments, the pharmaceutically acceptable carrier comprisesserum albumin.

The pharmaceutical composition of the invention is formulated to becompatible with its in-tended route of administration. Examples ofroutes of administration include parenteral, e.g., intrathecal,intra-arterial, intravenous, intradermal, subcutaneous, oral,transdermal (topical) and transmucosal administration.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine; propylene glycol or other syntheticsolvents; anti-bacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the injectable composition should be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequited particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a neu-regulin) in the required amount in an appropriatesolvent with one or a combination of ingre-dients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orStertes; a glidant such as colloidal silicon dioxide; a sweetening agentsuch as sucrose or saccharin; or a flavoring agent such as peppermint,methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the pharmaceutical compositions areformulated into ointments, salves, gels, or creams as generally known inthe art.

In certain embodiments, the pharmaceutical composition is formulated forsustained or controlled release of the active ingredient. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially (including liposomes targeted to infected cells withmonoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein includesphysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharma-ceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large thera-peutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of ad-ministration utilized. For any compoundused in the method of the invention, the therapeuti-cally effective dosecan be estimated initially from cell culture assays. A dose may beformu-lated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. The pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration.

Yet another aspect of the invention pertains to a method for inhibitinga neurological sensation mediated by sensory neurons in a subject, themethod comprising a step of inhibiting or reducing in a sensory neuronof the subject the acetylation of α-tubulin. Preferably the methodcomprises a step of introducing into the sensory neuron an inhibitor ofα-tubulin acetylation, preferably an inhibitor of α-tubulinacetyltransferase (Atat1).

In some embodiments the method of the invention may further comprise astep of administering to the subject an inhibitor of α-tubulinacetyltransferase.

Other embodiments of the method are provided which are for treating asubject suffering from a pathology associated with a neurologicalsensation, preferably wherein the pathology is pain such as acutemechanical pain, chronic mechanical pain, mechanical hyperalgesia,mechanical allodynia, inflammation, dental pain, cancer pain, visceralpain, arthritis pain, post-surgical pain, neuropathic pain, and laborpain.

Antagonists of the herein described invention are preferably selectedfrom the group of compounds consisting of inhibitory RNA, inhibitoryantibodies or fragments thereof, and/or small molecules. A detaileddescription of inhibitors and antagonists of acetylation of α-tubulin isprovided herein above.

In context of the invention it is also preferred that at least oneadditional therapeutic compound effective against pain, for example amorphine, an opioid or a non-opioid analgesic or other analgesic, isadministered to said subject.

The diseases treatable in context of the afore-described methods aredescribed herein above.

During the treatment or prevention it is preferred that at least oneadditional therapeutic effective against pain is administered to saidpatient, such as other analgesics, for example an opioid or a non-opioidanalgesic.

The above problem in the prior art is furthermore solved by a screeningmethod for identifying a compound useful for inhibiting a mechanicallyinduced, or chronical, neurological sensation comprising the steps of:

-   -   (a) Contacting (i) a biological cell expressing an α-tubulin        acetyltransferase gene, and/or (ii) a α-tubulin        acetyltransferase protein, with a candidate compound;    -   (b) Determining at least one of α-tubulin acetyltransferase        enzymatic activity or expression; and    -   (c) Comparing the activity, and/or the expression, as determined        in step (b) with the activity or expression of the α-tubulin        acetyltransferase in the absence of the candidate compound,        wherein a decrease between the measured activities and/or        expression of the α-tubulin acetyl-transferase indicates that        the candidate compound is an inhibitor of the α-tubulin        acetyltransferase and that the candidate compound is useful for        inhibiting a mechanically induced neurological sensation.

Some embodiments relate to the above method which is an ex vivo or invitro method.

The compound identified by the screening methodology of the invention ispreferably then suitable for use in the treatment of pain.

Candidate compounds for the screening are preferably selected from apolypeptide, peptide, glycoprotein, a peptidomimetic, an antibody orantibody-like molecule; a nucleic acid such as a DNA or RNA, for examplean antisense DNA or RNA, a ribozyme, an RNA or DNA aptamer, siRNA andthe like, including variants or derivatives thereof such as a peptidenucleic acid (PNA); a carbohydrate such as a polysaccharide oroligosaccharide and the like, including variants or derivatives thereof;a lipid such as a fatty acid and the like, including variants orderivatives thereof; or a small organic molecules including but notlimited to small molecule ligands, small cell-permeable molecules, andpeptidomimetic compounds.

Some embodiments of the screening method make use of assays, whereinexpression of the α-tubulin acetyltransferase is determined by detectingα-tubulin acetyltransferase mRNA, for example using a PCR based assay,or by detecting α-tubulin acetyltransferase protein, for exampleimmunologically. Assays for detecting or quantifying mRNA expression arewell known to those skilled in the art. Such assays could be qualitativeor quantitative, however, the latter is preferred.

In alternative or additional embodiments the α-tubulin acetyltransferaseactivity may be determined by measuring the α-tubulin acetylation. Inthis aspect the above screening method may be adapted from a screeningof biological cells, to a cell free assay system suitable to measureα-tubulin acetylation. In this embodiment the above screening method isaltered such that instead of a “biological cell” a “cell-free system” isprovided. A cell free system for assessing α-tubulin acetylation mayinclude as components isolated or recombinantly expressed α-tubulin, anacetylation donor such as acetyl-CoA, and an isolated or recombinantlyexpressed α-tubulin acetyltransferase. The amount of α-tubulinacetylation in the system in presence or absence of the candidatecompound is then determined by monitoring the reaction by eithermonitoring the decline of the donor molecule or the conversion ofα-tubulin to acetylated α-tubulin.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present invention will now be further described in the followingexamples with reference to the accompanying figures and sequences,nevertheless, without being limited thereto. For the purposes of thepresent invention, all references as cited herein are incorporated byreference in their entireties. In the Figures:

FIG. 1A is a bar chart summarising the results of a tape test to assaylow threshold mechanosensation. Atat1^(cKO) mice demonstratedsignificantly less response events over the 5 minute counting period(t-Test, P<0.05). FIG. 1B is a bar chart showing the cotton swabanalysis assaying low threshold mechanosensation. Atat1^(cKO) micedemonstrated significantly less response events then Atat1^(Control)counterparts (t-Test, P<0.01). FIG. 1C is a graph of von Frey thresholdsshowing the significantly lower response frequency in Atat1^(cKO)animals (RM ANOVA, Holm-Sidak method, P<0.001). FIG. 1D is a bar chartshowing latency to awareness of a clip attached to the base of the tail.Atat1^(cKO) animals take significantly longer to respond to the stimulus(t-Test, P<0.01). FIG. 1E is a bar chart showing that there are nosignificant differences in the responses recorded to noxious heatbetween Atat1^(cKO) and Atat1^(Control) animals (t-Test, P>0.05). FIG.1F is a bar chart showing that there are no significant differences inmotor performance as assayed using the Rotorod test (RM ANOVA,Holm-Sidak method, P>0.05). Error bars indicate s.e.m.

FIG. 2A shows responses (top) and stimulus-response function (bottom) ofslowly adapting mechanoreceptor fibers (SAM), FIG. 2B shows rapidlyadapting mechanoreceptor fibers (RAM), FIG. 2C shows D-hair afferents,FIG. 2D shows Aδ-mechanonociceptors (AM) and FIG. 2E show C-fibrenociceptors from αTAT1control and αTAT1cko mice (two-way ANOVA withpost-hoc Bonferroni's test, SAM: P<0.001; RAM: P<0.0001; D-hair:P<0.0001; AM: P<0.0001; C-fibre: P<0.0001). FIG. 2F shows Mean von Freythresholds for C fibre discharge (Mann-Whitney test, P<0.01). The numberof fibres recorded is indicated in parentheses in each panel. *P<0.05;**P<0.01; ***P<0.001; ****P<0.0001. Error bars indicate s.e.m.

FIG. 3A shows stacked histograms showing the proportion of differentmechanogated currents activated by neurite indentation in sensoryneurons from control Atat1^(Control) and Atat1^(cKO) mice (χ² test,P<0.05). NR, non-responsive to given displacement 512 nm. FIG. 3B showsrepresentative traces of RA currents elicited by increasing probedisplacement on soma of Atat1^(Control) and Atat1^(cKO) sensory neurons.FIG. 3C shows threshold of activation of RA currents was determined asmechanical stimulus that elicited a current ≥20 pA. Closed circlesindicate individual recorded cells. Note the marked increase in thedisplacement threshold in Atat1^(cKO) sensory neurons (Mann-Whitneytest, P<0.01). e FIG. 3D shows stimulus response curve of RA currentsevoked by increasing probe displacement. Genetic depletion of αTAT1 insensory neuron significantly reduced RA-currents amplitude (two-wayANOVA with post-hoc Bonferroni's test, P<0.0001). FIG. 3E shows stackedhistograms showing the proportions of different mechano-gated currentsobserved in Atat1^(cKO) sensory neurons transfected with EGFP, αTAT1-YFPor αTAT1-GGL-YFP cDNA. Transfection of wild-type αTAT1 rescued the lossof mechanosensitivity, while transfection of catalytically inactiveαTAT1 (αTAT1-GGL-YFP) failed to restore it in Atat1^(cKO) sensoryneurons (χ² test, EGFP versus αTAT1-YFP, P<0.05; EGFP versusαTAT1-GGL-YFP, P>0.05). FIG. 3F shows stacked histograms showing theproportions of different mechano-gated currents observed in Atat1^(cKO)sensory neurons transfected with EGFP, α-tubulin^(K40R)-IBES-YFP (K40R)or α-tubulin^(K40Q)-IRES-YFP (K40Q) cDNA. Transfection of acetylatedα-tubulin mimics (K40Q) but not non-acetylatable α-tubulin mutant (K40R)restored mechanosensitivity in Atat1^(cKO) sensory neurons (χ² test,EGFP versus K40Q, P<0.05; EGFP versus K40R, P>0.05). The number ofneurons recorded is indicated in parentheses in each panel. **P<0.01;****P<0.0001; Error bars indicate s.e.m.

FIG. 4A shows anti-acetylated-α-tubulin staining of Atat1^(Control)cultured DRG cells (corresponding surface plot below). Note theprominent sub-membrane localisation of acetylated tubulin (Scale bar 5μm). FIG. 4B shows anti α-tubulin staining of Atat1^(Control) culturedDRG cells (Scale bar 5 μm). FIG. 4C shows anti-acetylated-α-tubulinstaining of Atat1^(Control) MEFs (Scale bar 20 μm). Note the evendistribution of acetylated tubulin in this cell type. FIG. 4D shows antiα-tubulin staining of Atat1^(Control) MEFs (Scale bar 20 μm). FIG. 4Eshows immunohistochemical staining of nerve fibres within the saphenousnerve taken from Atat1^(Control) mice. Anti-acetylated-α-tubulinstaining is in green) and myelin basic protein (MBP) is in red (Scalebar 10 μm). Note the sub-membrane localisation of the anti-acetylatedtubulin stain. FIG. 4F shows a fluorescent image of free nerve endingsin a whole mount cornea preparation from a Avil-Cre::SNAP^(CaaX) mouse.Acetylated tubulin is in green) and membrane bound SNAP staining is inred (Scale bar 30 μm). Note the strong co-localisation of signals. FIG.4G shows a super-resolution image of an anti α-tubulin staining ofAtat1^(Control) cultured DRG colour coded by depth (red close toobjective, Scale bar 5 μm). FIG. 4H shows a super-resolution image of anAnti-α tubulin stain in Atat1^(cKO) cultured DRG (Scale bar 5 μm). FIG.4I is a graphical summary of AFM analysis showing the pressure requiredto indent the membrane to 200, 400 and 600 nm respectively, using ablunt ended cantilever in cultured DRG taken from Atat1^(Control) andAtat1^(cKO) mice. A significantly higher pressure is required to indentthe membranes of Atat1^(cKO) neurons over Atat1^(Control) cells(Mann-Whitney test, P<0.01). FIG. 4J is a graph showing the relativeshrinkage of axonal outgrowths from Atat1^(Control) and Atat1^(cKO) DRGloaded with calcein (2 μM) in response to a hyperosmotic shock overtime. Deletion of Atat1 leads to a significant decrease in thepercentage shrinking of axons relative to control samples (ANOVA onranks, multiple comparison Dunn's Method, P<0.05). FIG. 4K is an imagefrom a cultured DRG cell showing an overlay of C8 SIR tubulin labelledmicrotubules before (purple) and after (green) hyperosmotic shock. Notethe clear compression of the microtubule cytoskeleton after shrinking(Scale bar 10 μm). FIG. 4L is a bar chart summarising osmoticallyinduced microtubule compression in DRG neurons from Atat1^(Control),Atat1^(cKO), and Atat1^(cKO) neurons transfected with tubulin-K40Q.There is significantly less compression in Atat1^(cKO) thanAtat1^(Control) neurons, which is rescued by transfection oftubulin-K40Q (ANOVA on ranks, multiple comparison Dunn's Method,P<0.05). Error bars indicate s.e.m.

EXAMPLES

Materials and Methods

Animals and Behavioural Experiments

To study the effect on touch sensitivity of deleting the ATAT1 gene wecrossed ATAT1^(fl/+) mice¹⁷ with a peripheral nervous system specificCre line Avil^(cre/+) mouse line¹⁸ to obtain Avil-Cre::ATAT1^(fl/+)(control) and Avil-Cre::Atat1^(fl/+) (cKO) animals. Mice were genotypedas described previously^(18,19) and maintained at the EMBL Mouse BiologyUnit, Monterotondo, Italy, in accordance with Italian legislation (Art.9, 27 Jan. 1992, no 116) under license from the Italian Ministry ofHealth.

For the tape response assay mice were left to acclimate in plexiglasscontainers for 15 min. A 3 cm long by 1 cm wide piece of tape (Identitape) was then gently applied along the spinal column on the back of theanimal. The mice were then monitored for 5 min and the number ofbehavioural responses recorded. A response was recorded whenever a mouseattempted to remove the tape by scratching, biting or shaking.

For the cotton swab test, mice were placed in plexiglass boxes atop anelevated mesh base, and allowed to habituate for 30 min. A cotton swabwas then ‘puffed out’ by pulling with forceps to increase its size. Thisenlarged swab was then applied to the hind paw of the animal using agentle brushing manner, firstly to the right and subsequently the lefthind paw.

For von Frey testing, mice were placed inside an open topped plexiglasscontainer on an elevated mesh platform to acclimate for 1 h. A series ofvon Frey filaments (North Coast Medical, NC12775-99) with final force of0.02 g to 1 g were applied to the animal's hind paw alternating left andright paw and a yes/no paw withdrawal response was recorded.

For the Tail clip assay, an alligator clip, covered with rubber tubing(to reduce tissue damage) and calibrated to exert 400 g force wasattached to the base of the tail of Atat1^(Control) and Atat1^(cKO) miceAnimals were placed in plexiglass containers and the latency toawareness of the clip as indicated by biting, vocalization or graspingwas measured.

For the hot plate assay mice were placed on a hot plate (Ugo Basile,35150) pre-heated to 55° C. and latency time was measured until a jump,hind paw flick or hind paw lick were observed. In case of the lack ofany response the mice were removed from the hot plate after 30 s.

A modified version of the rotarod test was performed on naiveAtat1^(Control) and Atat1^(cKO) mice. Briefly, mice were habituated for5 mM to the stationary dowels of the rotarod (Rotarod 3375-5 TSEsystems). Each step, either habituation or test was followed by a 5minute rest period with food and water ad libitum. Mice were thenhabituated for 5 mM to the moving dowels at 5 RPM. Following this themice were tested at 5, 10, 15, 20 and 25 RPM respectively for 2 mM atrial. The time spent on the dowels was then calculated. A fall or twofull spins while gripping the dowel was considered a fail during thetest.

Ex Vivo Electrophysiology

The skin nerve preparation was used essentially as previouslydescribed²⁷. Briefly, mice were sacrificed using CO2 inhalation, and thesaphenous nerve together with the skin of the hind limb was dissectedfree and placed in an organ bath. The chamber was perfused with asynthetic interstitial fluid (SIF buffer) consisting of (in mM): NaCl,123; KCl, 3.5; MgSO4, 0.7; NaH2PO4, 1.7; CaCl2, 2.0; sodium gluconate,9.5; glucose, 5.5; sucrose, 7.5; and HEPES, 10 at a pH of 7.4.) The skinwas placed with the corium side up, and the nerve was placed in anadjacent chamber for fiber teasing and single-unit recording. Singleunits were isolated with a mechanical search stimulus applied with aglass rod and classified by conduction velocity, von Frey hairthresholds and adaptation properties to suprathreshold stimuli. Acomputer-controlled nanomotor (Kleindiek Nanotechnik) was used to applymechanical ramp-and-hold stimuli of known amplitude and velocity.Standardized displacement stimuli of 2 s or 10 s duration were appliedto the receptive field at regular intervals (interstimulus period, 30s). The probe was a stainless steel metal rod with a flat circularcontact area of 0.8 mm. The signal driving the movement of the linearmotor and raw electrophysiological data were collected with a Powerlab4.0 system and Labchart 7.1 software (AD instruments), Spikes werediscriminated off-line with the spike histogram extension of thesoftware.

Patch Clamping

DRG neurons were collected from mice and dissociated as described²⁷. Insome cases they were transfected using the Nucleofector system (LonzaAG) in 20 μl of Mouse Neuron Nucleofector solution from the SCNnucleofector kit (Lonza AG) and a total 4-5 μg of plasmid DNA at roomtemperature using the preinstalled program SCN Basic Neuro program 6.After electroporation, the cell suspension was transferred to 500 μl ofRPMI 1640 medium (Gibco) for 10 min at 37° C. This suspension,supplemented with 10% horse serum, was used to plate the cells ontoglass coverslips for recording. The RPMI medium supplemented with 100ng/ml nerve growth factor (NGF), 50 ng/ml BDNF was replaced with thestandard DRG medium 3-4 h later. Electrophysiology experiments began 12h after plating.

Whole-cell recordings from isolated DRG neurons were made as previouslydescribed²³. Recordings were made from DRG neurons using fire-polishedglass electrodes with a resistance of 3-7 Mg. Extracellular solutioncontained (mM): NaCl 140, MgCl2 1, CaCl2 2, KCl 4, glucose 4 and HEPES10 (pH 7.4), and electrodes were filled with a solution containing (mM):KCl 130, NaCl 10, MgCl2 1, EGTA 1 and HEPES 10 (pH 7.3). Cells wereperfused with drug-containing solutions by moving an array of outlets infront of the patched cells (WAS02; Ditel, Prague). Observations weremade with Observer A1 inverted microscope (Zeiss) equipped with a CCDcamera and the imaging software AxioVision. Membrane current and voltagewere amplified and acquired using EPC-10 amplifier sampled at 40 kHz;acquired traces were analyzed using Patchmaster and Fitmaster software(HEKA). Pipette and membrane capacitance were compensated using the autofunction of Pulse. For most of the experiments, to minimize the voltageerror, 70% of the series resistance was compensated and the membranevoltage was held at −60 mV with the voltage-clamp circuit. Afterestablishing whole-cell configuration, voltage-gated currents weremeasured using a standard series of voltage commands Briefly, theneurons were pre-pulsed to −120 mV for 150 ms and depolarized from −65to +55 mV in increments of 5 mV (40 ms test pulse duration). Next theamplifier was switched to current-clamp mode and current injection wasused to evoke action potentials. If the membrane capacitance andresistance changed more than 20% after the mechanical stimulus, the cellwas regarded as membrane damaged and the data discarded. Mechanicalstimuli were applied using a heat-polished glass pipette (tip diameter3-5 μm), driven by a MM3A Micromanipulator system (Kleindiek), andpositioned at an angle of 45 degrees to the surface of the dish. Theprobe was positioned near the neurite, moved forward in steps of 200-600nm for 500 ms and then withdrawn. For analysis of the kinetic propertiesof mechanically activated current, traces were fit with singleexponential functions using the Fitmaster software (HEKA). Data arepresented as mean±s.e.m.

Immunofluorescence and Staining

For microtubule staining in DRG cultures, cells were washed once withPBS, and then fixed for 15 mM in cytoskeleton buffer (CB) pH 6.3containing 3% paraformaldehyde, 0.25% triton and 0.2% glutaraldehyde atroom temperature. Cells were then washed 3 times with PBST (0.3%triton). Samples were then subsequently blocked with 5% normal goatserum (NGS) in PBS for 1 h at room temperature. Cells were then placedovernight at 4° C. with primary anti α-tubulin (1:1000) (Sigma-Aldrich,T9026) or anti-acetylated-α-tubulin (1:1000) (Sigma-Aldrich, T7451) inPBS. Cells were then washed with PBS and incubated for 1 h withfluorescently labelled secondary antibodies (1:1000) (Alexa Fluor 546Lifetechnologies) for 1 h at room temperature. All images were acquiredusing a 40× objective on a Leica SP5 confocal microscope. Processing ofimages and generation of surface plots were performed using ImageJImages were deconvoluted using Huygens software.

Actin filaments in DRG primary cultures were stained withAlexa488-phalloidin at 0.5 μg/ml (Lifetechnologies). Briefly cells werefixed with fresh 4% PFA (EM grade, TAAB) in cytoskeleton buffer (10 mMMES, 138 mM KCl, 3 mM MgCl, 2 mM EGTA) freshly added supplemented of 0.3M sucrose, permeabilized in 0.25% Triton-X-100 (Sigma-Aldrich), andblocked in 2% BSA (Sigma-Aldrich).

Immunostaining of saphenous nerves was performed on paraffin sectionsafter fixation with PFA. Following rehydration, antigen retrieval wasperformed with 10 mM sodium citrate (pH 6) at boiling temperature for 10mM. Subsequently, sections were permeabilized (0.3% Triton X-100),blocked (5% goat serum) and stained with anti-acetylated-α-tubulin(Sigma-Aldrich, T7451) and anti-myelin basic protein (Chemicon, MAB386).

For cornea staining, the eyes were removed and fixed for 1 h in 4% PFAat room temperature. The cornea was then dissected and permeabilizedwith PBS-Triton 0.03% for 30 mM. Following this, the cornea was immersedin PBS-Triton 0.03% containing 1 μM SNAP surface 546 (New EnglandBiolabs) for 30 min. The samples were then washed with PBS-Triton 0.03%for 20 min and subsequently blocked with 5% normal goat serum inPBS-Triton 0.03% for 30 mM. The tissue was then stained withanti-acetylated-α-tubulin (1:500) overnight. Samples were then washedwith PBS and a secondary antibody (Alexa Fluor 488 Life-technologies)was added for 5 h. The samples were again washed with PBS and stainedwith DAPI 10 mM. The cornea was then mounted on glass with 100% glyceroland imaged.

For whole mount axon outgrowth assays, individual DRG were extractedfrom mice and grown in Matrigel (Corning) for 7 days. Preparations werefixed with 4% PFA for 5 minutes and labelled with the primary antibodyPGP9.5 (1:200) overnight at 4° C. The samples were then labeled withsecondary antibodies (1:1000) Alexa Fluor 546 Lifetechnologies) for 1 hat room temperature. All images were acquired using a Leica LMD 7000.

SNAP-tag labelling was carried out by intradermal injection of thefinger in anaesthetized mice of 2 ∞M BG TMRstar substrate as describedpreviously²⁸. After five hours the animals were sacrificed and thesamples were mounted in 80% glycerol for imaging.

Electron Microscopy of Saphenous Nerve

Saphenous nerves were dissected and postfixed for 24 h with fresh 4%(w/v) PFA, 2.5% (w/v) Glutaraldehyde (TAAB) in 0.1 M Phosphate buffer at4 C. Following postfixation, the samples were incubated for 2 h with 1%(w/v) OsO4 supplemented with 1.5% (w/v) Potassium Ferrocyanide, sampleswere dehydrated in Ethanol and infiltrated with propylene oxide/Epon(Agar) (1:1) followed by resin embedding. Ultrathin sections were cut(Ultracut S, Leica), counter-stained with Uranyl Acetate and LeadCitrate and observed with a Transmission Electron Microscope (TEM) Jeol1010 equipped with a MSC 791 CCD camera (Gatan).

Microfluidics

DRG neurons were suspended in 1:1 Matrigel in 10% FBS DMEM and seededonto a two-chamber microfluidic chip (Xona Microfluidics, SD150). Axonswere allowed to grow across the microchannels connecting the twochambers for 3-5 days. On the day of the experiment, media in both thecell body and axon chambers was replaced with media with no serum for 3h. 1 μM mono-biotinylated NGF purified in house from eukaryotic cellswas coupled with 1 μM streptavidin conjugated quantum dots 655 (LifeTechnologies) for 30 min on ice, then diluted to 5 nM in imaging buffer(as above) and then used to replace the media in the axon chamber. A 25%volume difference was kept between the cell body and the axon chamber toavoid backflow from the axon to the cell body chamber. After 1 hincubation at 37° C. in 5% CO2, retrograde transport of NGF-Qdot655containing endosomes was imaged using a confocal Ultraview Vox (PerkinElmer) equipped with a 5% CO2 humidified chamber at 37° C. 100 s timelapses were recorded using 300 ms exposure time Images were analyzedwith Imaris software using the particle tracking function andautoregressive motion track generation setting.

Superresolution Microscopy

The cells were washed once with 3 ml of warm PBS. Subsequently, thecells were fixed and permeabilized for 2 min in cytoskeleton buffercontaining 0.3% Glutaraldehyde and 0.25% Triton X-100. Following this,the cells were fixed for 10 min in cytoskeleton buffer containing 2%Glutaraldehyde and treated for 7 min with 2 ml of 0.1% SodiumBorohydride (NaBH4) in PBS. Cells were then washed 3 times for 10 min inPBS. The cells were incubated with primary antibody for 30 min (mouseanti α-tubulin, Neomarker, 1:500) in PBS+2% BSA After washing 3 timesfor 10 min with PBS, the cells were transferred to the secondaryantibody (goat anti mouse Alexa 647, 1:500, Molecular Probes A21236) atroom temperature for 30 min. The cells were then washed 3 times with PBSfor 10 min and then mounted for PALM imaging. At the time of imagingcells were overlaid with PALM blinking buffer: 50 mM Tris pH 8.0, 10 mMNaCl, 10% Glucose, 100 U/ml Glucose Oxidase (Sigma-Aldrich), 40 ug/mlCatalase (Sigma-Aldrich).

The analysis of microtubule (MT) network morphology was done using theopen source software CellProfiler²⁹. The MT signal was enhanced by atop-hat filter and then binarised with the same manual threshold for allimages. Binary images were skeletonized using CellProfiler's “skelPE”algorithm and the resulting skeleton was subjected to branchpointdetection. As an approximation for MT network complexity we divided thenumber of branchpoints by the number of pixels in the skeleton.Moreover, we measured the local angular distribution of the MTs in orderto assess whether they run in parallel, or in a crossing manner (angularvariance). To this end, we subjected each pixel to a rotatingmorphological filter using a linear structural element with a length of11 pixels, and recorded at which angle we obtained a maximum response.The inventors computed the response for angles from 0 to 170 degrees atsteps of 10 degrees since there is no information on MT polarity. Nextwe measured the local circular variance³ of the MT orientations in asliding window with a diameter of 51 pixels, using angle doubling as itis commonly done for axial data³. The circular variance has a value 1 ifthe MTs in a given region are completely parallel and has smaller values(down to 0) if the MTs are oriented in various directions. Finally, wecomputed the average circular variance of all MT pixels in a given cell.If this value were close to 1 it would mean that locally, on a lengthscale of 51 pixels, the MTs are parallel in most of the cell.

Atomic Force Microscopy (AFM)

Force spectroscopy measurements were performed by using a NanoWizard AFM(JPK Instruments, Berlin, Germany) equipped with a fluid chamber(Biocell; JPK) for live cell analysis and an inverted optical microscope(Axiovert 200; Zeiss) for sample observation.

DRG cells were seeded on glass coverslip previously coated with a firstlayer of polylysine (500 μg/ml for about 1 h room temperature) and asecond layer of laminin (20 μg/ml for about 1 h at 37° C.). The cellswere then cultured for at least 15 h before measurements. Then, thesample was inserted into the fluid chamber (Biocell; JPK) immersed inculture medium and measurements were carried out at room temperature.The status of cells was constantly monitored by optical microscope.

Indenters for probing cell elasticity were prepared by mounting silicamicrospheres of 4.5 μm nominal diameter (Bangs Laboratories Inc.) totipless V-shaped silicon nitride cantilevers having nominal springconstants of 0.32 N/m or 0.08 N/m (NanoWorld, Innovative Technologies)by using UV sensitive glue (Loxeal UV Glue). Silica beads were pickedunder microscopy control. Before measurements the spring constant of thecantilevers was calibrated by using the thermal noise method.

By using the optical microscope the bead-mounted cantilever was broughtover the soma of single DRG and pressed down to indent the cell. Themotion of the z-piezo and the force were recorded. On each celleight-about ten force-distance curves were acquired with a force load of500 pN and at a rate of 5 μm sec-1 in closed loop feed-back mode.

Cell elastic properties were assessed by evaluating the Young's modulus(E) of the cell. This value was obtained by analyzing the approachingpart of the recorded F-D curves using the JPK DP software. The softwareconverts the approaching curve into force-indentation curves bysubtracting the cantilever bending from the signal height to calculateindentation. Afterwards force-indentation curves were fitted byHertz-Sneddon model for a spherical indenter according to this equation:

$F = {\frac{E}{1 - v^{2}}\left\lbrack {{\frac{a^{2} + R_{s}^{2}}{2}\ln\frac{R_{s} + a}{R_{s} - a}} - {aR}_{s}} \right\rbrack}$$\delta = {\frac{a}{2}\ln\frac{R_{s} + a}{R_{s} - a}}$

Here, δ is the indentation depth, a is the contact radius of theindenter, R is the silica bead radius, v is the sample's Poisson ratio(set to 0.5 for cell)³⁰ and E is Young's modulus. Fitting was performedat different indentations 200, 400 and 600 nm (see SI for examples offitting curves obtained).

For Young's modulus values, the statistical difference between twogroups of data was evaluated by using the non-parametric statisticalanalysis of the Mann-Whitney test (two-tailed distribution) by GraphPadPrism 5.0. A p value <0.05 was considered statistically significant.

Osmotic Shrinking Assays

Cultured DRG were loaded with 500 nM C8 SIR-Tubulin for 1 h at 37° C.and/or 2 μM calcein dye (Invitrogen C3100MP) for 30 mM in DRG at 37° C.The cells were then transferred to imaging buffer (10 mM Hepes pH 7.4,140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM D-glucose) at 320mOsm. Following a 5 mM acclimatization period the cells were subjectedto a 440 mOsm (osmolarity adjusted with mannitol) hyperosmotic shock for3 min. All imaging was carried out using a Leica SP5 resonant scanner.

Example 1 Conditional Atat1 Knock-Out Mice Have Impaired Sensation ofMechanical Innoxious and Noxious Stimuli

To investigate cell autonomous effects of Atat1 disruption in sensoryneurons the inventors took a conditional gene deletion strategy.Atat1^(fl) mice¹⁸ were crossed with a sensory neuron specific Cre driverline Avil-Cre¹⁹ to generate Avil-Cre::Atat1^(fl/fl) (referred to asAtat1^(cKO)) and control Avil-Cre::Atat1^(fl/+) mice (referred to asAtat1^(Control)). Mice were then subjected to a series of behaviouralassays. The inventors first tested their ability to detect an innocuousmechanical stimulus applied to the hairy skin. Adhesive tape was fixedgently to the backs of animals and the number of responses counted overa 5 minute observation period. While control mice made regular attemptsto remove the tape, Atat1^(cKO) mice effectively ignored it for much ofthe time, and the total number of responses was significantly lower(FIG. 1 a ).

The inventors next investigated the sensitivity of mice to innocuousmechanical stimuli applied to the glabrous skin by lightly stroking theunderside of the paw with a diffuse cotton swab²⁰. Again, Atat1^(cKO)mice responded significantly less to this stimulus than Atat1^(Control)mice (FIG. 1 b ). The inventors also examined whether mechanicalsensitivity to punctate stimuli was altered in Atat1^(cKO) mice byapplying von Frey filaments of calibrated forces to the hindpaw of mice.Control animals responded to forces as low as 0.07 g with a linearincrease in detection into the noxious range. However, Atat1^(cKO) micerequired significantly higher forces to evoke a response throughout therange of von-Frey filaments (FIG. 1 c ).

To investigate noxious mechanical sensitivity in more detail, theinventors analysed responses to a clip applied to the base of the tail.Atat1^(cKO) mice displayed substantially longer latencies to awarenessof the clip compared to Atat1^(Control) mice and again, essentiallyignored it for much of the time (FIG. 1 d ).

The inventors further tested whether noxious thermal detection waseffected by Atat1 deletion by measuring the time to response on ahotplate. The inventors observed no difference in withdrawal latenciesto noxious temperatures between Atat1^(cKO) and Atat1^(Control) mice(FIG. 1 e ). Finally the inventors assessed the motor coordination ofAtat1^(cKO) mice by evaluating their performance on a rotorod device.Atat1^(cKO) and Atat1^(Control) mice displayed statistically similarlatencies to fall from the rotating drum across all speeds tested. Thus,Atat1 is required for the detection of innocuous and noxious mechanicaltouch but not for noxious heat or proprioceptive coordination.

Example 2 Sensory Neuron Electrophysiological Responses are Impaired inAtat1 Conditional Knockout Mice

Sensory neuron axons terminate in the skin and form a diverse range offunctionally distinct mechanoreceptors that underlie the sense oftouch¹. They can be classified by their conduction velocity (into Aβ, Aδand C fibres), their adaptation properties (into rapidly adapting orslowly adapting) and by their mechanical thresholds. To determine howAtat1 deletion affects each of these populations and regulates touchsensitivity, the inventors utilized an ex vivo skin-nerve preparation torecord from single cutaneous sensory neurons in the saphenous nerve. Theinventors first considered fast conducting Aβ fibres, separating theminto slowly adapting (SAM) and rapidly adapting (RAM) mechanoreceptors.The inventors observed a striking reduction in the mechanicalsensitivity of SAM fibres that was apparent as a reduced number ofaction potentials per stimulus indentation (FIG. 2 a ) and a ˜10-foldincrease in the latency of the response in Atat1^(cKO) mice. Reductionsin firing frequencies were evident during both the ramp phase of themechanical stimulus and during the static phase. RAM fibres displayed asimilar reduction in their stimulus response function (FIG. 2 b ) and anincreased latency to the highest displacement stimulus. A characteristicof these fibres is that they display higher firing frequencies withincreasing stimulus speed²¹, a feature which was also reduced inAtat1^(cKO) mice. Mechanical, electrical thresholds and conductionvelocities were unchanged in Aβ fibres in the absence of Atat1. Theinventors next examined Aδ fibres which can be classified as D-hair andAδ-mechanonociceptors (AM) units by their mechanical threshold andadaptation properties. Both populations of mechanoreceptor displayedsignificant reductions in their stimulus response function (FIGS. 2 cand d ), longer latencies for mechanical activation, and decreasedsensitivity to dynamic stimuli. Electrical thresholds and conductionvelocities were unchanged in the absence of Atat1. Finally the inventorsconsidered C-fibres, the largest population of sensory afferents.Similar to all other fibre types, C-fibres exhibited a reduced number ofaction potentials evoked by indentation stimuli (FIG. 2 e ), and nochange in electrical properties or conduction velocity. Strikingly,mechanical thresholds of C-fibres were also significantly elevated inAtat1^(cKO) mice (FIG. 2 f ). Thus Atat1 is required for mechanicalsensitivity across all major fibre types innervating the skin.

Example 3 Modulation of Atat1 Enzymatic Activity RegulatesMechanosensitivity

To determine how deletion of Atat1 influences mechanotransduction insensory neurons the inventors recorded mechanosensitive currents fromcultured DRG neurons indented with a blunt glass probe. Such a stimuluscan evoke mechanically gated currents in ˜90% of DRG neurons that arefurther classified as rapidly adapting (RA), intermediate-adapting (IA)and slowly adapting (SA) responses²³. In the absence of Atat1, theinventors observed a marked loss in the number of mechanically sensitiveneurons in the DRG that was evident across each subtype of current (FIG.3 a ). Furthermore, the small proportion of neurons which stilldisplayed mechanosensitive currents in Atat1^(cKO) mice exhibitedsignificantly reduced current amplitudes and higher thresholds (FIG. 3b-d ), but no difference in their activation kinetics. Other functionalparameters such as voltage gated channel activity, resting membranepotential, action potential threshold, and pH sensitivity wereindistinguishable between Atat1^(Control) and Atat1^(cKO) miceindicating that the reduced mechanical sensitivity of DRG neurons doesnot arise from compromised membrane properties.

The inventors next asked whether the reduction in mechanosensitivityobserved in Atat1^(cKO) mice is dependent upon the α-tubulinacetyltransferase activity of αTAT1 by testing if mechanically activatedcurrents could be re-established by expression of exogenous cDNAs. As apositive control the inventors determined that transfection of anAtat1-YFP construct rescued mechanosensitivity in Atat1^(cKO) culturesand that the proportion of RA, IA and SA responses across the DRGreturned to control levels (FIG. 3 e ). The inventors subsequentlytransfected a catalytically inactive form of αTAT1 that has noacetyltransferase activity but remains functional²⁴ (termed αTAT1-GGL).Expression of αTAT1-GGL did not restore mechanosensitivity inAtat1^(cKO) neurons, and the inventors observed no difference in theproportion of mechanically activated current types compared to mock eGFPtransfection (FIG. 3 e ). Atat1 has also been demonstrated to acetylateother substrates in addition to α-tubulin²⁵. Therefore, to determinewhether α-tubulin acetylation underlies the mechanosensory phenotype inAtat1^(cKO) mice, the inventors transfected a K40Q point mutant ofα-tubulin that genetically mimics α-tubulin lysine 40 acetylation.Expression of K40Q α-tubulin rescued mechanosensitivity of Atat1^(cKO)DRG neurons to Atat1^(Control) levels, while a charge conserving controlmutation (K40R) had no significant effect (FIG. 3 f ). Collectivelythese data indicated that the acetyltransferase activity of αTAT1regulates mechanosensitivity and that acetylated α-tubulin is the likelyeffector.

Example 4 Microtubule Organization in Peripheral Sensory Neurons

The inventors investigated a potential structural contribution ofacetylated tubulin to mechanosensitivity by examining the distributionof acetylated microtubules in sensory neurons. Strikingly, the inventorsobserved that acetylated α-tubulin was concentrated in a prominent banddirectly under the plasma membrane in cultured DRG neurons (FIG. 4 a ),while total α-tubulin was distributed evenly across the cytoplasm of allcells (FIG. 4 b ). Importantly, this band was not present innon-mechanosensory cells such as fibroblasts where acetylated α-tubulinwas present throughout the microtubule network (FIGS. 4 c and d ). Theinventors further examined the distribution of acetylated α-tubulin inintact preparations of the peripheral nervous system. Again, acetylationwas highly enriched under the membrane of axons in the saphenous nerve(FIG. 4 e ) and also at sensory neuron terminal endings in the cornea(FIG. 4 f ).

The loss of the acetylated α-tubulin sub-membrane band in Atat1^(cKO)mice could potentially impact upon the organization of microtubules inDRG neurons and thereby influence mechanosensitivity. Indeed, it hasbeen recently shown that the arrangement of microtubules is importantfor mechanosensitivity of hypothalamic osmosensory neurons²⁶. Utilizingsuper-resolution microscopy and automated analysis of α-tubulindistribution, the inventors were unable however to detect any differencein the spatial arrangement of microtubules in sensory neurons fromAtat1^(Control) and Atat1^(cKO) mice (FIGS. 4 g and h ). Furthermore,the organization of the actin cytoskeleton also appeared unaltered inAtat1^(cKO) mice.

What then is the function of the acetylated α-tubulin band, and how doesit impact upon mechanosensitivity across the range of mechanoreceptorsin the skin? One possibility is that it sets the rigidity of cellsthereby influencing the amount of force required to displace the plasmamembrane and activate mechanosensitive channels. The inventors exploredthis by directly measuring membrane elasticity using atomic forcemicroscopy. In DRG neurons from Atat1^(cKO) mice the inventors observedthat cellular stiffness was significantly higher across a range ofindentations extending from displacements that perturbed mainly themembrane (200 nm) to those that deformed the underlying cytoskeleton(600 nm) (FIG. 4 i ). Thus higher forces are required to indent sensoryneurons from Atat1^(cKO) mice than Atat1^(Control) mice. The inventorsinvestigated this further by assaying the sensitivity of neurons tohyperosmotic induced shrinkage. In the absence of Atat1, sensory neuronaxons displayed less shrinkage than their control counterparts, aneffect that could be rescued by expression of the acetylation mimickingmutation α-tubulin K40Q (FIG. 4 j ).

Finally the inventors examined how the microtubule cytoskeleton respondsto compression induced by osmotic pressure. Using a novel tubulinlabelling fluorescent dye the inventors were able to resolve individualmicrotubule bundles in live imaging experiments. Strikingly, in DRGneurons from Atat1^(cKO) mice the inventors observed significantlyreduced microtubule displacement upon application of hyperosmoticsolutions (FIGS. 4 k and 4 l ), again supporting the premise that in theabsence of α-tubulin acetylation sensory neurons are more resistant tomechanical deformation.

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The invention claimed is:
 1. A method for reducing expression of ATAT1in a subject experiencing mechanical pain mediated by sensory neurons,the method comprising: administering to the subject an effective amountof a nucleic acid that is capable of reducing expression of ATAT1;wherein the mechanical pain is selected from the group consisting of:inflammatory pain, acute mechanical pain, chronic mechanical pain,mechanical hyperalgesia, mechanical allodynia, visceral pain, and laborpain.
 2. The method of claim 1, wherein the acute mechanical pain is dueto physical trauma selected from soft tissue damage, infection andinflammation.
 3. The method of claim 1, wherein the acute mechanicalpain is selected from pain due to surgery, cuts, bruises, fractured orbroken bones, hemorrhoids, intestinal gas, dyspepsia and dental pain. 4.The method of claim 1, wherein the chronic mechanical pain is a painthat lasts longer than 1 month or beyond the resolution of an acutetissue injury or is recurring or is associated with tissue injury and/orchronic diseases that are expected to continue or progress.
 5. Themethod of claim 1, wherein the chronic mechanical pain is selected frompain due to inflammatory disease, cancer, arthritis, chronic wounds,cardiovascular incidents, spinal cord disorders, central nervous systemdisorder, recovery from surgery, and neuropathy.
 6. The method of claim1, wherein the chronic mechanical pain is selected from pain due toosteoarthritis, rheumatoid arthritis, fibromyalgia, meralgiaparesthetica, back pain, angina, carpel tunnel syndrome, menstruation,and hemorrhoids.
 7. The method of claim 1, wherein the visceral pain isselected from pain due to menstruation, inflammation, cancer, dyspepsia,and intestinal gas.
 8. The method of claim 1, wherein the nucleic acidis capable of reducing expression of ATAT1 through RNA interference. 9.The method of claim 8, wherein the nucleic acid encodes a shRNA.
 10. Themethod of claim 8, wherein the nucleic acid encodes a siRNA.
 11. Themethod of claim 1, wherein mechanical pain is reduced.
 12. A method forreducing pain in a subject experiencing mechanical pain mediated bysensory neurons, the method comprising: administering to the subject aneffective amount of a nucleic acid that is capable of reducingexpression of ATAT1; wherein the mechanical pain is selected from thegroup consisting of: inflammatory pain, acute mechanical pain, chronicmechanical pain, mechanical hyperalgesia, mechanical allodynia, visceralpain, and labor pain.
 13. The method of claim 12, wherein the acutemechanical pain is due to physical trauma selected from soft tissuedamage, infection, and inflammation.
 14. The method of claim 12, whereinthe acute mechanical pain is selected from pain due to surgery, cuts,bruises, fractured or broken bones, hemorrhoids, intestinal gas,dyspepsia and dental pain.
 15. The method of claim 12, wherein thechronic mechanical pain is a pain that lasts longer than 1 month orbeyond the resolution of an acute tissue injury or is recurring or isassociated with tissue injury and/or chronic diseases that are expectedto continue or progress.
 16. The method of claim 12, wherein the chronicmechanical pain is selected from pain due to inflammatory disease,cancer, arthritis, chronic wounds, cardiovascular incidents, spinal corddisorders, central nervous system disorder, recovery from surgery, andneuropathy.
 17. The method of claim 12, wherein the chronic mechanicalpain is selected from pain due to osteoarthritis, rheumatoid arthritis,fibromyalgia, meralgia paresthetica, back pain, angina, carpel tunnelsyndrome, menstruation, and hemorrhoids.
 18. The method of claim 12,wherein the visceral pain is selected from pain due to menstruation,inflammation, cancer, dyspepsia, and intestinal gas.
 19. The method ofclaim 12, wherein the nucleic acid is capable of reducing expression ofATAT1 through RNA interference.
 20. The method of claim 19, wherein thenucleic acid encodes a shRNA.
 21. The method of claim 19, wherein thenucleic acid encodes a siRNA.
 22. The method of claim 12, whereinmechanical pain is reduced.